L. van der Straat, J.A. Tamayo-Ramos, T. Schonewille and L.H. de Graaff. Overexpression of a modified 6-phosphofructo-1-kinase results in an increased itaconic acid productivity in Aspergillus niger. AMB Express, 2013,3:57
50
CHAPTER 4
Abstract
A modified 6-phosphofructo-1-kinase was expressed in a citrate producing Aspergillus niger strain in combination with cis-aconitate decarboxylase from Aspergillus terreus to study the effect on the production of itaconic acid. The modified pfkA gene was also expressed in combination with the itaconic acid biosynthetic cluster from A. terreus, which consists of cis-aconitate decarboxylase cadA, a putative mitochondrial transporter mttA and a putative plasmamembrane transporter mfsA. The combined expression of pfkA and cadA resulted in increased citrate levels, but did not show increased itaconic acid levels. The combined expression of pfkA with the itaconic acid biosynthetic cluster resulted in significantly increased itaconic acid production at earlier time points. Also the itaconic acid productivity increased significantly. The maximum itaconic acid productivity that was reached under these conditions was 0.15 g/L/h, which is only a factor 17 lower than the 2.5 g/L/h that according to the US Department of Energy should be achieved to have an economically feasible production process.
Keywords
Itaconic acid, Aspergillus niger, modified 6-phosphofructo-1-kinase, cis-aconitate decarboxylase cadA, mitochondrial transporter mttA, plasmamembrane transporter mfsA.
51
OVEREXPRESSION OF A MODIFIED PKF RESULTS IN AN INCREASED ITACONIC ACID PRODUCTIVITY IN A. NIGER
4
Introduction
The bio-based production of chemicals from second-generation plant biomass is both economically and scientifically challenging. The cost-efficient production of chemicals requires highly efficient production processes based on highly optimized production organisms. One of the promising production organisms in this respect is Aspergillus niger.
This filamentous fungus is industrially used for the production of plant cell wall hydrolyzing enzymes and for the production of metabolites like citrate. Based on its current use, A.
niger can relatively easily be modified for the production of bio-based chemicals. Itaconic acid is a metabolic derivative of citric acid and therefore can potentially be produced in A.
niger. Itaconic acid (methyl succinic acid) is a C5 dicarboxylic acid. It is one of the top twelve building block chemicals that can be produced from plant biomass sugars via a fermentative process. 1 The methylene group of itaconic acid can participate in polymerization reactions and on the basis of this characteristic itaconic acid can be used for the production of synthetic polymers. 2 It is further used as a bioactive compound in agriculture, pharmacy and as a medicine. 3 Itaconic acid can also be used as a starting compound for enzymatic transformations to form useful poly-functional building blocks. 4
Nowadays, Aspergillus terreus is mostly used for the commercial production of itaconic acid in a submerged fermentation process. 3, 5 The metabolic pathway for the production of itaconic acid is similar to the metabolic pathway of citric acid production in A. niger.
Given this similarity in biosynthesis in A. niger we have started to develop an itaconic acid production process based on A. niger citric acid producing strains.
Citric acid is commercially produced using A. niger reaching production levels of more than 200 g/L, while A. terreus reaches itaconic acid production levels of 80 g/L. 6 Therefore A.
niger is the host of choice for our research. A. niger is not able to produce itaconic acid naturally since it lacks the essential gene cadA encoding cis-aconitate decarboxylase. The cadA gene was identified via a clone-based transcriptomics 7 approach and via an enzyme purification 8, 9 approach. In the genome of A. terreus two putative transporters flank the cadA gene.
Recently it was shown that especially the putative mitochondrial transporter mttA is crucial for an efficient itaconic acid production process in A. niger and that, to a minor extent, the putative plasmamembrane transporter mfsA also has a positive effect on itaconic acid production. 9, 10 In the past, several research groups tried to increase the flux through the glycolytic pathway since that might greatly enhance the productivity of acid production. The work of Schreferl-Kunar in 1989 already showed that A. niger mutants that were selected on the basis of increased citric acid secretion levels have strongly increased hexokinase and 6-phoshofructo-1-kinase activity. 11 However, overexpression of 6-phosphofructo-1-kinase in a citric acid producing A. niger strain did not lead to increased citric acid levels. 12 Nonetheless, cultivation of A. niger on high sugar concentrations showed that the control
52
CHAPTER 4
over the glycolytic flux at the level of 6-phosphofructo-1-kinase was absent under these conditions. 13 Apparently mutations in the 6-phosphofructo-1-kinase gene led to an enzyme that was less inhibited by citrate. This was confirmed by Mlakar and Legiša (2006) who showed that of the two described A. niger 6-phosphofructo-1-kinases 14 one form was moderately inhibited by citrate while the other form proved to be completely resistant to citrate inhibition. 15 Later on, a specific truncated version of the pfkA gene from A. niger, resistant to citrate inhibition but still highly active, was obtained. 16 The expression of this modified 6-phosphofructo-1-kinase gene in A. terreus resulted in increased itaconic acid production levels. 17
In this manuscript, we report the effect of overexpressing the modified 6-phosphofructo-1-kinase in A. niger on organic acid production, when combined with the cadA gene from A. terreus as well as in combination with the itaconic acid biosynthetic cluster cadA, mttA and mfsA.